Topic A) Ca2+ influx channels (35% effort) Project 1: The role of tether proteins in assembly of the ER/PM junctions at PI(4,5)P2-rich microdomains that determines STIM1 conformation and Orai1 gating. The Orai1 channel is the pore forming subunit that is activated by the ER Ca2+ sensor STIM1. Ca2+ influx by Orai1 mediates virtually all cellular functions from gene activation to exocytosis on time scales of msec to days. However, aberrant over activation of Orai1 and likely the TRPC channels occurs in many diseases and most often is the trigger of the disease as occurs in pancreatitis and Sjgren's syndrome. The activity of these channels is tightly regulated by the entering Ca2+ itself and is mediated by the STIM1 regulator SARAF. In an early work we identified Extended Synaptotagmin 1 (E-Syt1) as one of the proteins that tether the ER/PM junction. In continuation of these studies preliminary data show that a more critical protein in the junction is ANO8. A readout of proteins function at the ER/PM junction is how the protein called SARAF causes Ca2+-dependent inactivation of the Orai1-mediated Ca2+ influx. SARAF do so by binding to STIM1 only in PI(4,5)P2-rich ER/PM junction. The slow Ca2+-dependent inactivation (SCDI) of Orai1 by SARAF binding to STIM1 serves as a probe of the conformation and microdomain localization of the Orai1-STIM1 complex. In a related project, we knockout Orai1 in mice and discovered a novel function for Orai1-mediated acinar cells secretion. Ca2+ influx by Orai1 control secretion of antibacterial agents by the pancreas that are essential for gut control of the microbiome. Without this secretion, the microbiome changed resulting in bacteremia, sepsis and death. Thus, manipulation of Orai1 activity cannot be used to treat diseases of secretory glands. However, these new findings are directing our work to understand how the pancreas and the salivary glands control the gut and oral cavity, respectively, microbiomes in health and disease states. Future plans for STIM1, Orai1 and TRPC channels: a) We continue to examine how STIM1 gates the TRPC and Orai1 channels. At this time, we are studying the role of ANO8 and other potential tethers in forming the PI(4,5)P2-rich and PI(4,5)P2-poor domains and how they affect channel gating. b) We are analyzing mice with targeted deletion of Orai1 in the pancreas and salivary glands to understand how the pancreas and the salivary glands control the gut and oral cavity, respectively, microbiomes in health and disease states. Topic B) Intracellular Ca2+ channels (15% effort) Project 2: Regulation of the lysosomal two-pore channels and Ca2+ signaling by TMEM63a. Lysosomal Ca2+ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca2+ signaling was the discovery of the two-pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated channels regulated by mTORC1, but not by NAADP. In a previous study, we provided conclusive evidence that TPC2 is NAADP-activated channel and further discovered that TPC2 is a Mg2+ and energy sensor. More recently, in collaboration with Dr. Freichel, U of Heidelberg we discovered a new TRPML1-like protein that expressed in the lysosomes and, more important, in secretory granules of acinar cells, that regulate Ca2+ release from the organelles. We are in the process of understanding the physiological function of this protein. Future plans for TRPML and TPC channels: We are completing the studies on the role of TRPML1-L1 in organelle Ca2+ signaling and regulated exocytosis by a) using the TRPML1-L1-/- mice to determine their possible role in vivo; b) In collaboration with a former postdoctoral fellow, we are characterizing the role of TRPML1 in bone metabolism through its role in osteoclast function and osteopetrosis. Topic C) fluid and HCO3- secretion (35% effort) Project 3: Intracellular Cl- as a Signaling ion that Potently Regulate HCO3- Transporters. Introduction & Significance: A major topic in the lab is studying the transport function and role of members of the SLC26 transporters superfamily, CFTR and the Na+-HCO3- cotransporters NBCs. In addition to the transport of HCO3-, many of the transporters are transport Cl- or are exposed to large changes in Cl- concentration during fluid and electrolyte transport. An example, are the ducts of secretory glands such as the salivary glands and the pancreas, in which Cl-in is reduced from 40-50 mM in the resting state to 5-10 mM in the stimulated secretory state. How cells sense and respond to changes in Cl-in is not known. We discovered that Cl-in functions as a signaling ion by regulation the activity of the basolateral 2Na+-1HCO3- cotransporter NBCe1-B and likely other Cl- and HCO3- transporters. We identified two Cl- binding sites in the N terminus of NBCe1-B that mediates the regulation. NBCe1-B is regulated by Cl- only when it is fully activated by IRBIT. IRBIT is a multifunctional protein that in resting cells it is sequestered by the IP3 receptors and inhibit Ca2+ signaling. Cell stimulation releases IRBIT from the IP3Rs and becomes available for regulation of the HCO3- transporters. Since Cl- is a major anion in mammalian cells involved in transport processes that determines the intracellular activity of many ions and plasma membrane potential. Our findings that Cl-in functions as a regulator of cellular Na+ and HCO3- concentrations and transepithelial transport through modulating the activity of several electrogenic Na+-HCO3- transporters has several physiological implications. At the present we continue to determine the mode by which Cl-in regulate the transporters. Preliminary studies show that regulation of NBCe1-B by Cl-in is modulated by the phosphatases PP1 and Calcineurin and the kinases SPAK and CaMKII, respectively. Future plans for fluid and HCO3- secretion: a) We are studying the regulation of other transporters by Cl-; b) We are studying how the phosphatases PP1 and Calcineurin and the kinases SPAK and CaMKII, respectively modulate regulation of NBCe1-B by Cl-in; c) We continue the project to determine if FDA approved CFTR potentiators and correctors correct the aberrant fluid secretion in mouse models of Sjgren's syndrome and pancreatitis.